The CDC recently released a report detailing antibiotic resistant threats in the US. Of particular emphasis in the CDC report is the increased prevalence of multidrug-resistant, Gram-negative bacteria (MDR- GNB) and the need to develop the next generation of antibiotics to combat them. All Gram-negative bacteria rely on a set of homologous, yet highly-specific, outer membrane TonB-dependent transporters (TBDTs) to import critical nutrients from their environment, especially metals like iron, which are bound by high-affinity, metal chelating compounds called siderophores. Recent antibiotic developments have shown that siderophore-antibiotic conjugates can be selectively targeted to specific bacteria, and that this delivery mechanism overcomes several key antibiotic resistance mechanisms. However, a significant limitation of this delivery system is the low expression levels of the TBDTs. The long-term objective of this proposal is to provide a mechanistic understanding of how Gram-negative bacteria transcriptionally regulate their TBDTs in order to manipulate this process to selectively up-regulate TBDT levels and enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. In this proposal we will elucidate the structural basis for protein interaction events that are responsible for up-regulatin the transcription of particular TBDTs. As a model system we are using the pseudobactin BN7/8 transport system from Psuedomonas putida that consists of the TBDT, PupB, the inner membrane ?-regulator, PupR, and the cytoplasmic ?-factor, PupI. To accomplish our objective we will pursue the following two specific aims: 1) delineate the mechanism by which the PupR:PupI interaction at the cytoplasmic face of the inner membrane is altered to allow transcriptional activation by PupI, and 2) establish the thermodynamics and atomic-level structural details of the interaction between the PupB and PupR. For the successful completion of our aims we will employ a multidisciplinary approach including NMR spectroscopy, X-ray crystallography, molecular biology, cellular assays, and biophysical techniques such as isothermal titration calorimetry. This research will provide the first structural information for a ?-regulator, explain how localization of a ?-factor to the inner membrane limits its activity, and th extent to which periplasmic interactions between the TBDT and ?-regulator lead to conformational changes that might be important for controlling transcriptional activation.